Subsarcolemmal Mitochondria Research Articles

Subsarcolemmal and intermyofibrillar mitochondria proteome differences disclose functional specializations in skeletal muscle

Skeletal muscle is a highly specialized tissue that contains two distinct mitochondria subpopulations, the subsarcolemmal (SS) and the intermyofibrillar (IMF) mitochondria. Although it is established that these mitochondrial subpopulations differ functionally in several ways, limited information exists about the proteomic differences underlying these functional differences. Therefore, the objective of this study was to biochemically characterize the SS and IMF mitochondria isolated from rat red gastrocnemius skeletal muscle. We separated the two mitochondrial subpopulations from skeletal muscle using a refined method that provides an excellent division of these unique mitochondrial subpopulations. Using proteomics of mitochondria and its subfractions (intermembrane space, matrix and inner membrane), a total of 325 distinct proteins were identified, most of which belong to the functional clusters of oxidative phosphorylation, metabolism and signal transduction. Although more gel spots were observed in SS mitochondria, 38 of the identified proteins were differentially expressed between the SS and IMF subpopulations. Compared to the SS mitochondrial, IMF mitochondria expressed a higher level of proteins associated with oxidative phosphorylation. This observation, coupled with the finding of a higher respiratory chain complex activity in IMF mitochondria, suggests a specialization of IMF mitochondria toward energy production for contractile activity.

Characteristics of the rat cardiac sphingolipid pool in two mitochondrial subpopulations

Mitochondrial sphingolipids play a diverse role in normal cardiac function and diseases, yet a precise quantification of cardiac mitochondrial sphingolipids has never been performed. Therefore, rat heart interfibrillary mitochondria (IFM) and subsarcolemmal mitochondria (SSM) were isolated, lipids extracted, and sphingolipids quantified by LC–tandem mass spectrometry. Results showed that sphingomyelin (∼10,000pmol/mg protein) was the predominant sphingolipid regardless of mitochondrial subpopulation, and measurable amounts of ceramide (∼70pmol/mg protein) sphingosine, and sphinganine were also found in IFM and SSM. Both mitochondrial populations contained similar quantities of sphingolipids except for ceramide which was much higher in SSM. Analysis of sphingolipid isoforms revealed ten different sphingomyelins and six ceramides that differed from 16- to 24-carbon units in their acyl side chains. Sub-fractionation experiments further showed that sphingolipids are a constituent part of the inner mitochondrial membrane. Furthermore, inner membrane ceramide levels were 32% lower versus whole mitochondria (45pmol/mg protein). Three ceramide isotypes (C20-, C22-, and C24-ceramide) accounted for the lower amounts. The concentrations of the ceramides present in the inner membranes of SSM and IFM differed greatly. Overall, mitochondrial sphingolipid content reflected levels seen in cardiac tissue, but the specific ceramide distribution distinguished IFM and SSM from each other.

Isolating the segment of the mitochondrial electron transport chain responsible for mitochondrial damage during cardiac ischemia

Ischemia damages the mitochondrial electron transport chain (ETC), mediated in part by damage generated by the mitochondria themselves. Mitochondrial damage resulting from ischemia, in turn, leads to cardiac injury during reperfusion. The goal of the present study was to localize the segment of the ETC that produces the ischemic mitochondrial damage. We tested if blockade of the proximal ETC at complex I differed from blockade distal in the chain at cytochrome oxidase. Isolated rabbit hearts were perfused for 15 min followed by 30 min stop-flow ischemia at 37 °C. Amobarbital (2.5 mM) or azide (5 mM) was used to block proximal (complex I) or distal (cytochrome oxidase) sites in the ETC. Time control hearts were buffer-perfused for 45 min. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated. Ischemia decreased cytochrome c content in SSM but not in IFM compared to time control. Blockade of electron transport at complex I preserved the cytochrome c content in SSM. In contrast, blockade of electron transport at cytochrome oxidase with azide did not retain cytochrome c in SSM during ischemia. Since blockade of electron transport at complex III also prevented cytochrome c loss during ischemia, the specific site that elicits mitochondrial damage during ischemia is likely located in the segment between complex III and cytochrome oxidase.

Mitochondrial impairment contributes to cocaine-induced cardiac dysfunction: Prevention by the targeted antioxidant MitoQ

The goal of this study was to assess mitochondrial function and ROS production in an experimental model of cocaine-induced cardiac dysfunction. We hypothesized that cocaine abuse may lead to altered mitochondrial function that in turn may cause left ventricular dysfunction. Seven days of cocaine administration to rats led to an increased oxygen consumption detected in cardiac fibers, specifically through complex I and complex III. ROS levels were increased, specifically in interfibrillar mitochondria. In parallel there was a decrease in ATP synthesis, whereas no difference was observed in subsarcolemmal mitochondria. This uncoupling effect on oxidative phosphorylation was not detectable after short-term exposure to cocaine, suggesting that these mitochondrial abnormalities were a late rather than a primary event in the pathological response to cocaine. MitoQ, a mitochondrial-targeted antioxidant, was shown to completely prevent these mitochondrial abnormalities as well as cardiac dysfunction characterized here by a diastolic dysfunction studied with a conductance catheter to obtain pressure–volume data. Taken together, these results extend previous studies and demonstrate that cocaine-induced cardiac dysfunction may be due to a mitochondrial defect.

Mitochondrial fission and remodelling contributes to muscle atrophy

Mitochondria are crucial organelles in the production of energy and in the control of signalling cascades. A machinery of pro-fusion and fission proteins regulates their morphology and subcellular localization. In muscle this results in an orderly pattern of intermyofibrillar and subsarcolemmal mitochondria. Muscular atrophy is a genetically controlled process involving the activation of the autophagy-lysosome and the ubiquitin-proteasome systems. Whether and how the mitochondria are involved in muscular atrophy is unknown. Here, we show that the mitochondria are removed through autophagy system and that changes in mitochondrial network occur in atrophying muscles. Expression of the fission machinery is per se sufficient to cause muscle wasting in adult animals, by triggering organelle dysfunction and AMPK activation. Conversely, inhibition of the mitochondrial fission inhibits muscle loss during fasting and after FoxO3 overexpression. Mitochondrial-dependent muscle atrophy requires AMPK activation as inhibition of AMPK restores muscle size in myofibres with altered mitochondria. Thus, disruption of the mitochondrial network is an essential amplificatory loop of the muscular atrophy programme.

Skeletal muscle apoptosis following 7 days of denervation‐induced muscle disuse

Chronic decreases in skeletal muscle contractile activity result in muscle atrophy, reduced force production and increased fatigue. However, the mechanisms of disuse‐induced activation of apoptotic signaling cascades which lead to atrophy are not fully established. To study these pathways C57BL/6 (WT) and Bax/Bak knockout (KO) animals were denervated for 7 days. This model of muscle disuse resulted in a decrease in mitochondrial respiration and a 2‐fold increase in the production of reactive oxygen species. Activity of the anti‐oxidant enzyme catalase was elevated by 2.5‐fold in subsarcolemmal (SS) mitochondria from WT and KO animals. Denervation resulted in higher cofilin levels within SS mitochondria, suggesting an increased rate of cofilin translocation from the cytosol. This translocation of cofilin may have contributed to the approximate 2‐fold increase in cleaved Poly‐ADP ribose polymerase (Parp) following disuse. Denervation also resulted in a 1.7‐fold increase in DNA fragmentation in WT animals, but no fragmentation was evident in the KO group. These data suggest that: 1) Bax and Bak contribute to the degree of DNA fragmentation following denervation and 2) chronic muscle disuse increases oxidative stress which stimulates the translocation of pro‐apoptotic proteins, such as cofilin, to promote protein release, and contribute to the loss of muscle mass. This work was supported by NSERC.

Mitochondria‐specific overexpression of phospholipid hydroperoxide glutathione peroxidase (GPx4) attenuates ischemia/reperfusion (I/R) associated apoptosis

A primary determinant of the extent of I/R injury in the mitochondrion is antioxidant defense capacity. Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a unique antioxidant enzyme capable of reducing peroxidized acyl groups in phospholipids, providing protection to biological membranes. The goal of this study was to evaluate the impact of mitochondria‐specific overexpression of PHGPx on apoptotic susceptibility following cardiac I/R. Transgenic mice were created in which PHGPx was overexpressed in the mitochondrion (mPHGPx). MPHGPx and control hearts were subjected to global no‐flow ischemia (20 min) followed by reperfusion (90 min). MPHGPx subsarcolemmal mitochondria (SSM) displayeda decreased propensity for undergoing apoptosis as compared to I/R controls, with no differences in interfibrillar mitochondria (IFM). Caspase‐3 and −9 activities were decreased in mPHGPx SSM relative to I/R control (P<0.05), with no differences in IFM. Mitochondrial permeability transition poreopening was decreased in mPHGPx SSM as compared to control(P<0.05), with no difference in IFM. Cyclophilin D was significantly decreased in mPHGPx SSM relative to I/R control (P<0.05), with no differences in IFM. These results indicate that mitochondria‐specific PHGPx overexpression protects SSM against I/R‐associated apoptosis.(Supported by NIH DP2 DK083095, AHA 0815406D, AHA 0855484D, NIH T32 HL090610)

Uncoupling and Inward Migration of Subsarcolemmal Mitochondria in Rat Heart during Early Diabetes

Myocyte loss is an established feature in hearts of both individuals with diabetes mellitus and in several animal models of the disease. Studies attribute this to an increase in apoptosis resulting from elevation in cytoplasmic cytochrome c and activation of caspase-3. To date, spatial, structural and functional changes subsarcolemmal mitochondria (SSM), which protect myocytes from circulating insults, undergo during early diabetes remains poorly characterized. Using the streptozotocin-induced diabetic rat model we show that after 5-6 weeks of diabetes, SSM disaggregate and migrate inwards. Diabetic SSM (dSSM) also exhibited increased biogenesis, were smaller with more compact cristae, possessed higher citrate synthase activity, produced more reactive oxygen species (ROS), increased interaction with sarcoplasmic reticulum (SR), and took up more Ca2+ . Atomic force microscopy also revealed that forty percent of dSSM also possessed a circumferential “ribbon-like” structure and 12% of these were leaky. dSSM also contained 65% less superoxide dismutase-I and 66% less connexin 43, a protein that regulates the activity of mitochondria K ATP channels and opening of the mitochondrial permeability transition pore. Insulin-treatment blunted these changes. The inward migration of SSM during diabetes is likely to leave myocytes vulnerable to plasmalemmal Ca2+ spikes resulting from the barrage of circulating agonists. Persistent increases in ROS production and lower connexin 43 content are also likely to trigger leaking of dSSM and elevate cytoplasmic levels of cytochrome c and apoptosis-inducing factors. Thus, we propose that compensatory changes to ensure adequate ATP production and maintenance of ionic homeostasis during diabetes switches SSM from protecting myocytes to inducing their demise. (This work was funded in part by grants from NIH to WGM and KRB and AHA to MCZ).

Increased subsarcolemmal lipids in type 2 diabetes: effect of training on localization of lipids, mitochondria, and glycogen in sedentary human skeletal muscle

The purpose of the study was to investigate the effect of aerobic training and type 2 diabetes on intramyocellular localization of lipids, mitochondria, and glycogen. Obese type 2 diabetic patients (n = 12) and matched obese controls (n = 12) participated in aerobic cycling training for 10 wk. Endurance-trained athletes (n = 15) were included for comparison. Insulin action was determined by euglycemic-hyperinsulinemic clamp. Intramyocellular contents of lipids, mitochondria, and glycogen at different subcellular compartments were assessed by transmission electron microscopy in biopsies obtained from vastus lateralis muscle. Type 2 diabetic patients were more insulin resistant than obese controls and had threefold higher volume of subsarcolemmal (SS) lipids compared with obese controls and endurance-trained subjects. No difference was found in intermyofibrillar lipids. Importantly, following aerobic training, this excess SS lipid volume was lowered by approximately 50%, approaching the levels observed in the nondiabetic subjects. A strong inverse association between insulin sensitivity and SS lipid volume was found (r(2)=0.62, P = 0.002). The volume density and localization of mitochondria and glycogen were the same in type 2 diabetic patients and control subjects, and showed in parallel with improved insulin sensitivity a similar increase in response to training, however, with a more pronounced increase in SS mitochondria and SS glycogen than in other localizations. In conclusion, this study, estimating intramyocellular localization of lipids, mitochondria, and glycogen, indicates that type 2 diabetic patients may be exposed to increased levels of SS lipids. Thus consideration of cell compartmentation may advance the understanding of the role of lipids in muscle function and type 2 diabetes.

The protective effect of Ro -54864 on ischemia-reperfusion myocardial function and mitochondrial

Objective To investigate the protective effect of Ro -54864 on ischemia reperfusion myocardial function and mitochondrial in the isolated perfused Langendorff model. Methods After equilibration for 20 min, the hearts were randomly allocated into 5 groups as follows. Sham group: perfusion for 115 min in a Krebs-Henseleit (K-H) buffer. Control group: global ischemia for 25 min followed by reperfusion for 90 min. Ro group: administration of 0.1 μmol/L Ro-54864 K-H buffer for 10 min before ischemia; ATR group: administration of 20 μmol/L atractyloside K-H buffer for 10 min before ischemia; ATR+Ro group: administration of 0.1 μmol/L Ro-54864 and 20 μmol/L atractyloside K-H buffer for 10 min before ischemia. Separate experiments were performed to determine homodynamic variables after equilibration for 20 min, before ischemia, after reperfusion for 15 min, and 60 min. At the end of reperfusion, infarct size was determined with 2,3, 5-triphenyltetrazolium chloride(TTC) staining. And the subsarcolemmal mitochondria (SSM) was isolated and its morphology was observed and quantified under the electron microscope. Results After reperfusion for 15 min and 60 min, homodynamic variables and CF in Ro group were higher than the other groups, Ro-54864 decreased infarct size significantly (28%±3% vs. 41%±3% in control hearts; P＜0.05), and myocardial mitochondrial damage in Ro Group reduced significantly than that of the control group. However, the outcome of ATR group were adverse, and those parameters in ATR+Ro group were equivalent with respect to the Control group. Conclusion The peripheral benzodiazepine receptor, Ro -54864, salvages myocardium from infarction and protectes ischemic heart mitochondria and improves functional recovery. Nonetheless, the mitochondrial permeability transition pore opener, atractyloside, deteriorated ischemic myocardial injury, reduce the protective effect of Ro54864 on ischemic myocardial. Key words: Peripheral Benzodiazepine Receptor; Ro-54864; Atractyloside; Mitochondrial Permeability Transition

Enhanced apoptotic propensity in diabetic cardiac mitochondria: influence of subcellular spatial location

Cardiovascular complications, such as diabetic cardiomyopathy, account for the majority of deaths associated with diabetes mellitus. Mitochondria are particularly susceptible to the damaging effects of diabetes mellitus and have been implicated in the pathogenesis of diabetic cardiomyopathy. Cardiac mitochondria consist of two spatially distinct subpopulations, termed subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM). The goal of this study was to determine whether subcellular spatial location is associated with apoptotic propensity of cardiac mitochondrial subpopulations during diabetic insult. Swiss Webster mice were subjected to intraperitoneal injection of streptozotocin or citrate saline vehicle. Ten weeks following injection, diabetic hearts displayed increased caspase-3 and caspase-9 activities, indicating enhanced apoptotic signaling (P < 0.05, for both). Mitochondrial size (forward scatter) and internal complexity (side scatter) were decreased in diabetic IFM (P < 0.05, for both) but not in diabetic SSM. Mitochondrial membrane potential (Delta(Psim)) was lower in diabetic IFM (P < 0.01) but not in diabetic SSM. Mitochondrial permeability transition pore (mPTP) opening was increased in diabetic compared with control IFM (P < 0.05), whereas no differences were observed in diabetic compared with control SSM. Examination of mPTP constituents revealed increases in cyclophilin D in diabetic IFM. Furthermore, diabetic IFM possessed lower cytochrome c and BcL-2 levels and increased Bax levels (P < 0.05, for all 3). No significant changes in these proteins were observed in diabetic SSM compared with control. These results indicate that diabetes mellitus is associated with an enhanced apoptotic propensity in IFM, suggesting a differential apoptotic susceptibility of distinct mitochondrial subpopulations based upon subcellular location.

Validation of 99mTc-labeled “4+1” fatty acids for myocardial metabolism and flow imaging

Our group has synthesized technetium-labeled fatty acids (FA) that are extracted into the myocardium and sequestered due to heart-type fatty acid binding protein (H-FABP) binding. In this article, we further address the detailed subcellular distribution and potential myocardial metabolism of [(99m)Tc]"4+1" FA. Experiments were conducted using isolated hearts of Wistar rats, as well as of wild-type and H-FABP(-/-) mice. Myocardium samples underwent subcellular fractionation [subsarcolemmal mitochondria (SM), intermyofibrillar mitochondria (IM), cytosol with microsomes, and nuclei and crude membranes] and analysis by thin-layer chromatography and high-performance liquid chromatography. The largest fraction of tissue radioactivity was associated with cytosol [79.69+/-8.88% of infused dose]. About 9.07+/-0.95% and 3.43+/-1.38% of the infused dose were associated with SM and IM fractions, respectively. In the rat heart, etomoxir, an inhibitor of carnitin-palmitoyl transferase I, did not significantly decrease radioactivity associated with mitochondrial fractions, whereas myocardial extraction of [(123)I]-labeled 15-(p-iodophenyl)-pentadecanoic acid (13.26% vs. 49.49% in controls) and the radioactivity associated with the SM and IM fractions were blunted. The percentage of the infused dose in the mitochondrial and crude fractions increased with the number of NH-amide groups of the FA derivative. Absence of H-FABP significantly decreased radioactivity count in the cytosolic fraction (P<.001). No metabolic product of [(99m)Tc]"4+1" FA could be detected in any isolated heart. Myocardial [(99m)Tc]"4+1" FA extraction reflects binding to H-FABP and membrane structures (including the mitochondrial membrane). However, the compounds do not undergo mitochondrial metabolism because they do not reach the mitochondrial matrix.

Reply to “Letter to the editor: ‘Does a reduction in ADP-limited respiration indicate impaired mitochondrial function?’”

LETTERS TO THE EDITORReply to “Letter to the editor: ‘Does a reduction in ADP-limited respiration indicate impaired mitochondrial function?’”Erinne R. Dabkowski and John M. HollanderErinne R. Dabkowski and John M. HollanderPublished Online:01 Aug 2009https://doi.org/10.1152/ajpheart.00539.2009MoreSectionsPDF (29 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat reply: We would like to address the letter by Schwarzer and Doenst (4), regarding our recent study (2) investigating the differential influence of streptozotocin-induced diabetes mellitus on interfibrillar and subsarcolemmal mitochondria in mice. This study identified a multitude of differential responses associated with the diabetic phenotype, which included cardiac contractile abnormalities, mitochondrial morphological changes, increases in reactive oxygen species, enhanced oxidative stress, changes in a specific cardiolipin species, and impaired “resting” mitochondrial respiratory rates (1).We are in agreement with Drs. Schwarzer and Doenst (4) that the inclusion of state 3 respiration analyses would have strengthened our study and enabled a more conclusive interpretation of the data on state 4 respiration. This is not to say that an examination of state 4 respiration independent of state 3 respiration is entirely without value. In fact, in a similar model of diabetes mellitus (streptozotocin), it was shown that decreases in state 3 respiration can occur concomitantly with decreases in state 4 respiration (3).We would also like to address the second point made by Schwarzer and Doenst (4), regarding the inclusion of two different methodologies for assessing mitochondrial function. In this regard, it is important to point out that at no time do we make comparisons between different electron transport chain complexes (i.e., complex I vs. complex III, etc.). Rather, we only make comparisons of electron transport chain function between treatment groups (i.e., control vs. diabetic, subsarcolemmal mitochondria vs. interfibrillar mitochondria, etc.) in which the same methodology is being employed. Thus the utilization of different methods for assessment should not confound the interpretation of the findings for comparisons made solely between treatment groups.In conclusion, we hope that our response will help to clarify the interpretation of the data presented regarding the mitochondrial functional parameters presented in Dabkowski et al. (2).REFERENCES1 Chance B, Williams GR. The respiratory chain and oxidative phosphorylation. Adv Enzymol Relat Subj Biochem 17: 65–134, 1956.PubMed | Google Scholar2 Dabkowski ER, Williamson CL, Bukowski VC, Chapman RS, Leonard SS, Peer CJ, Callery PS, Hollander JM. Diabetic cardiomyopathy-associated dysfunction in spatially distinct mitochondrial subpopulations. Am J Physiol Heart Circ Physiol 296: H359–H369, 2009.Link | ISI | Google Scholar3 King KL, Young ME, Kerner J, Huang H, O'Shea KM, Alexson SE, Hoppel CL, Stanley WC. Diabetes or peroxisome proliferator-activated receptor alpha agonist increases mitochondrial thioesterase I activity in heart. J Lipid Res 48: 1511–1517, 2007.Crossref | PubMed | ISI | Google Scholar4 Schwarzer M, Doenst T. Letter to the editor: “Does a reduction in ADP-limited respiration indicate impaired mitochondrial function?” Am J Physiol Heart Circ Physiol. doi:10.1152/ajpheart.00434.2009.Link | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: J. M. Hollander, West Virginia Univ. School of Medicine, Div. of Exercise Physiology, Center for Interdisciplinary Research in Cardiovascular Sciences, 1 Medical Center Dr., Morgantown, WV 26506 (e-mail: [email protected]) Download PDF Previous Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 297Issue 2August 2009Pages H890-H890 Copyright & PermissionsCopyright © 2009 the American Physiological Societyhttps://doi.org/10.1152/ajpheart.00539.2009History Published online 1 August 2009 Published in print 1 August 2009 Metrics

Letter to the editor: “Does a reduction in ADP-limited respiration indicate impaired mitochondrial function?”

to the editor: In a recent study, Dabkowski et al. ([7][1]) investigated the differential influence of streptozotocin-induced diabetes mellitus on interfibrillar and subsarcolemmal mitochondria in mice. They found decreased mitochondrial size, increased superoxide production, increased oxidative

The role of PGC-1α on mitochondrial function and apoptotic susceptibility in muscle

Mitochondria are critical for cellular bioenergetics, and they mediate apoptosis within cells. We used whole body peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) knockout (KO) animals to investigate its role on organelle function, apoptotic signaling, and cytochrome-c oxidase activity, an indicator of mitochondrial content, in muscle and other tissues (brain, liver, and pancreas). Lack of PGC-1alpha reduced mitochondrial content in all muscles (17-44%; P < 0.05) but had no effect in brain, liver, and pancreas. However, the tissue expression of proteins involved in mitochondrial DNA maintenance [transcription factor A (Tfam)], import (Tim23), and remodeling [mitofusin 2 (Mfn2) and dynamin-related protein 1 (Drp1)] did not parallel the decrease in mitochondrial content in PGC-1alpha KO animals. These proteins remained unchanged or were upregulated (P < 0.05) in the highly oxidative heart, indicating a change in mitochondrial composition. A change in muscle organelle composition was also evident from the alterations in subsarcolemmal and intermyofibrillar mitochondrial respiration, which was impaired in the absence of PGC-1alpha. However, endurance-trained KO animals did not exhibit reduced mitochondrial respiration. Mitochondrial reactive oxygen species (ROS) production was not affected by the lack of PGC-1alpha, but subsarcolemmal mitochondria from PGC-1alpha KO animals released a greater amount of cytochrome c than in WT animals following exogenous ROS treatment. Our results indicate that the lack of PGC-1alpha results in 1) a muscle type-specific suppression of mitochondrial content that depends on basal oxidative capacity, 2) an alteration in mitochondrial composition, 3) impaired mitochondrial respiratory function that can be improved by training, and 4) a greater basal protein release from subsarcolemmal mitochondria, indicating an enhanced mitochondrial apoptotic susceptibility.

Exercise training induces a cardioprotective phenotype and alterations in cardiac subsarcolemmal and intermyofibrillar mitochondrial proteins

Endurance exercise is known to provide cardioprotection against ischemia-reperfusion-induced myocardial injury, and mitochondrial adaptations may play a critical role in this protection. To investigate exercise-induced changes in mitochondrial proteins, we compared the proteome of subsarcolemmal and intermyofibrillar mitochondria isolated from the myocardium of sedentary (control) and exercise-trained Sprague-Dawley rats. To achieve this goal, we utilized isobaric tags for relative and absolute quantitation, which allows simultaneous identification and quantification of proteins between multiple samples. This approach identified a total of 222 cardiac mitochondrial proteins. Importantly, repeated bouts of endurance exercise resulted in significant alterations in 11 proteins within intermyofibrillar mitochondria (seven increased; four decreased) compared with sedentary control animals. Furthermore, exercise training resulted in significant changes in two proteins within subsarcolemmal mitochondria (one increased; one decreased) compared with sedentary control animals. Differentially expressed proteins could be classified into seven functional groups, and several novel and potentially important cardioprotective mediators were identified. We conclude that endurance exercise induces alterations in mitochondrial proteome that may contribute to cardioprotective phenotype. Importantly, based on our findings, pharmacological or other interventions could be used to develop a strategy of protecting the myocardium during an ischemic attack.

Distinct Mitochondrial Subpopulation Response in a Type 2 Diabetic Model

Mitochondrial dysfunction plays a critical role in the pathogenesis of type 2 diabetes mellitus. Examination of mitochondria is complicated by the fact that two spatially distinct mitochondria populations exist in the cardiac myocyte, interfibrillar mitochondria (IFM) which situate between the myofibrils, and subsarcolemmal mitochondria (SSM) which exist beneath the sarcolemma. The goal of this study was to determine whether spatially distinct cardiac mitochondrial subpopulations are differentially affected by type 2 diabetic insult. Db/db mice were sacrificed at 20 weeks of age and mitochondrial subpopulations isolated. State 3 respiration was significantly lower in db/db SSM contributing to decreased RCR ratios (P&lt;0.05). Electron transport chain complex activities (I, III, IV) were significantly decreased only in diabetic SSM as compared to control (P&lt;0.05 for all three). Uncoupling protein 3 was significantly increased only in db/db SSM (P&lt;0.05). Manganese superoxide dismutase (MnSOD) protein was significantly elevated in db/db IFM suggesting a potential spatially‐specific protective mechanism. Mitochondrial heat shock protein 70 (mthsp70), a mitochondrial import protein, was significantly decreased only in db/db SSM. These findings suggest that type 2 diabetic insult impacts spatially distinct cardiac mitochondria differently with SSM displaying greater dysfunctional profiles.

Skeletal Muscle Mitochondrial Subpopulation Response in a Type 2 Diabetic Mouse Model

Dysfunctional mitochondria are central to the pathogenesis of type 2 diabetes mellitus. In skeletal muscle, two spatially distinct mitochondrial subpopulations exist. The interfibrillar mitochondria (IFM) which are situated between myofibrils and the subsarcolemmal mitochondria (SSM) which reside directly below the sarcolemma. The objective of this study was to determine how type 2 diabetes mellitus differentially affects distinct mitochondrial subpopulations in skeletal muscle. Db/db mice and littermate controls were sacrificed at 20 weeks of age and mitochondrial subpopulations were isolated from the gastrocnemius muscle. State 3 respiration in db/db mice was significantly lower in both IFM and SSM populations compared to respective controls leading to a decrease in RCR ratios (P&lt;0.05). Electron transport chain complex I and IV activities were significantly decreased in the diabetic IFM with no change in the SSM (P&lt;0.05 for both). Superoxide production was increased in the diabetic IFM and SSM, although to a greater extent in the IFM. These findings indicate that both mitochondrial subpopulations in mouse gastrocnemius muscle are affected during type 2 diabetes mellitus, however, the IFM exhibit a greater dysfunctional profile.

Muscle plasticity of Inuit sled dogs in Greenland

This study examined flexible adjustments of skeletal muscle size, fiber structure, and capillarization in Inuit sled dogs responding to seasonal changes in temperature, exercise and food supply. Inuit dogs pull sleds in winter and are fed regularly throughout this working season. In summer, they remain chained to rocks without exercise, receiving food intermittently and often fasting for several days. We studied two dog teams in Northern Greenland (Qaanaaq) where dogs are still draught animals vital to Inuit hunters, and one dog team in Western Greenland (Qeqertarsuaq) where this traditional role has been lost. Northern Greenland dogs receive more and higher quality food than those in Western Greenland. We used ultrasonography for repeated muscle size measurements on the same individuals, and transmission electron microscopy on micro-biopsies for summer-winter comparisons of muscle histology, also within individuals. At both study sites, dogs' muscles were significantly thinner in summer than in winter - atrophy attributable to reduced fiber diameter. Sarcomeres from West Greenland dogs showed serious myofilament depletion and expansion of the sarcoplasmatic space between myofibrils during summer. At both study sites, summer samples showed fewer interfibrillar and subsarcolemmal mitochondria, and fewer lipid droplets between myofibrils, than did winter samples. In summer, capillary density was higher and inter-capillary distance smaller than in winter, but the capillary-to-fiber-ratio and number of capillaries associated with single myofibers were constant. Increased capillary density was probably a by-product of differential tissue responses to condition changes rather than a functional adaptation, because thinning of muscle fibers in summer was not accompanied by reduction in the capillary network. Thus, skeletal muscle of Inuit dogs responds flexibly to changes in functional demands. This flexibility is based on differential changes in functional components: mitochondrial numbers, lipid droplet size, and the number of contractile filaments all increase with increasing workload and food supply while the capillary network remains unchanged.

In obese rat muscle transport of palmitate is increased and is channeled to triacylglycerol storage despite an increase in mitochondrial palmitate oxidation

Intramuscular triacylglycerol (IMTG) accumulation in obesity has been attributed to increased fatty acid transport and/or to alterations in mitochondrial fatty acid oxidation. Alternatively, an imbalance in these two processes may channel fatty acids into storage. Therefore, in red and white muscles of lean and obese Zucker rats, we examined whether the increase in IMTG accumulation was attributable to an increased rate of fatty acid transport rather than alterations in subsarcolemmal (SS) or intermyofibrillar (IMF) mitochondrial fatty acid oxidation. In obese animals selected parameters were upregulated, including palmitate transport (red: +100%; white: +51%), plasmalemmal FAT/CD36 (red: +116%; white: +115%; not plasmalemmal FABPpm, FATP1, or FATP4), IMTG concentrations (red: approximately 2-fold; white: approximately 4-fold), and mitochondrial content (red +30%). Selected mitochondrial parameters were also greater in obese animals, namely, palmitate oxidation (SS red: +91%; SS white: +26%; not IMF mitochondria), FAT/CD36 (SS: +65%; IMF: +65%), citrate synthase (SS: +19%), and beta-hydroxyacyl-CoA dehydrogenase activities (SS: +20%); carnitine palmitoyltransferase-I activity did not differ. A comparison of lean and obese rat muscles revealed that the rate of change in IMTG concentration was eightfold greater than that of fatty acid oxidation (SS mitochondria), when both parameters were expressed relative to fatty transport. Thus fatty acid transport, esterification, and oxidation (SS mitochondria) are upregulated in muscles of obese Zucker rats, with these effects being most pronounced in red muscle. The additional fatty acid taken up is channeled primarily to esterification, suggesting that upregulation in fatty acid transport as opposed to altered fatty acid oxidation is the major determinant of intramuscular lipid accumulation.